We propose to identify genetic loci required for invasive hyphal growth in Aspergillus fumigatus. This ubiquitous fungus is the leading causative agent of invasive aspergillosis, a global disease that particularly threatens individuals with leukemia, lung disease, cancer, or solid organ transplantation. Despite aggressive antifungal therapy, the overall mortality rate of invasive aspergillosis is greater than 50%. The most unifying clinical feature among those afflicted individuals is destruction of lung tissue and blood vessels by Aspergillus hyphal invasion. Because complete avoidance of this ubiquitous fungus is nearly impossible, prevention or restriction of Aspergillus invasive hyphal growth is essential in controlling invasive aspergillosis. In the proper environment, spores of A. fumigatus send out long tubular cells known as hyphae and grow by tip extension searching for nutrients. When A. fumigatus spores enter the respiratory system of susceptible hosts through inhalation, they form highly polarized hyphae, invade the lungs and other organs, and cause destruction of these host tissues. We hypothesize that invasive hyphal growth, which enables A. fumigatus to efficiently probe potential food sources in solid media in the environment, is also an important characteristic for the fungus to invade human tissues and cause invasive aspergillosis. Molecules and biochemical pathways required for invasive hyphal growth, therefore, represent promising antifungal targets. However, the molecular details of underlying A. fumigatus invasive hyphal growth have remained unclarified. We propose to use forward genetic approaches to systematically uncover genetic loci that regulate A. fumigatus invasive hyphal growth. We will: (1) Generate additional 30,000 random insertional A. fumigatus mutants;(2) Screen the mutant collection in vitro for defective invasive hyphal growth and identify mutated genes in selected mutants using inverse PCR and sequencing. (3) Confirm a subset of candidate genes involved in invasive hyphal growth by target gene deletion and complementation. We have recently developed an efficient and simplified method to generate a large number of random insertional mutants of A. fumigatus via Agrobacterium-mediated-transformation. A pilot study using 8000 mutants generated by this method indicates that our proposed aims can be accomplished. Although it is beyond the scope of this proposal, all the promising genetic loci identified will be confirmed by target gene deletion and complementation, and their roles in pathogenesis will be examined in the future using animal models. Our proposed research will functionally uncover genetic components that govern invasive hyphal growth in this important human pathogen. This project is a stepping stone for the development of a more comprehensive understanding of growth characteristics that enable A. fumigatus, a saprophytic fungus living on compost, to cause invasive diseases in humans. PUBLIC HEALTH RELEVANCE: Aspergillus fumigatus is the leading causative agent of invasive aspergillosis, an often fatal disease in immuno-compromised or immuno-suppressed individuals. This fungus causes invasive mycoses through hyphal invasion of host tissue and organs. The proposed research will identify genes that allow A. fumigatus to grow invasively in the hyphal form, a growth characteristic important for its pathogenicity. Information obtained from the proposed research will be harnessed to develop novel mechanisms to control invasive aspergillosis and allow for the discovery of promising new antifungal drug targets. .